In 1898, an Austrian microbiologist Heinrich Winterberg made a curious observation: the number of microbial cells in his samples did not match the number of colonies formed on nutrient media (Winterberg 1898). Within a decade, this mismatch was quantified, and turned out to be surprisingly large: the percent of cultivable cells proved to be less than 1%. This signified one of the earliest steps towards an important discovery known today as the Great Plate Count Anomaly (Staley and Konopka 1985), - arguably the oldest unresolved microbiological phenomenon. This proposal addresses this phenomenon in its clinical manifestation. Microorganisms from the human microbiome, including the oral cavity, are poorly cultivable: over 50% of oral microflora are still missing from culture collections. This is a significant impediment to the study of human health because oral bacteria, including uncultivated species, have been implicated in a variety of systemic diseases. Here we propose two approaches to domesticate previously uncultivated species from the human oral cavity. Both approaches represent a substantial departure from standard cultivation techniques, and address some of their limitations. We successfully tested these approaches in our environmental research on uncultivated microorganisms from aquatic environment and soils, and adapt them here to address clinical needs. In Aim 1, we employ a long-term, single cell cultivation method. Single cell cultivation eliminates competition from other strains and prevents overgrowing by weed species, whereas long-term (weeks) incubation provides appropriate conditions for slower growing organisms. Collectively, this gives a chance to grow (and be detected) to any cell, no matter how slowly dividing or rare. To account for additional growth preferences, we developed two modifications of the method, based on liquid and solid media. It is encouraging that our recent application of this method to environmental microorganisms led to isolation of several remarkably novel species. In Aim 2, we utilize the observation that, in nature, microbial species do not grow in isolation but often as codependent components of microbial consortia. The ecological interactions between microbial partners have many aspects, from co-metabolism of a carbon source, to exchange of metabolites, to interspecies signaling. Such interactions, and the resulting co-dependence in growth, may make some species unable to divide if deprived of their natural synergies. We developed a method for co-cultivating microorganisms, and successfully applied it to cultivating novel aquatic and soil species. We also designed a simple technique to separate the microbial consortia into their component species. This enables investigation of the benefits provided by the partner species, with the goal of mimicking the synergistic requirements by supplementing the medium with the compound(s) exchanged within consortia. This possibility is explored in Aim 3. Aim 3 is the natural product chemistry component of the study. We will interrogate microbial consortia from Aim 2, chosen for their importance to clinical research, and investigate the chemical underpinning of growth codependence in microbial co-cultures. Using bioassay-guided fractionations, coupled with state-of-the-art microscale LC-MS and NMR analyses, we will determine the chemical structure of factors responsible for growth induction/stimulation within consortia. We will then supplement standard media with these factors, making them capable of supporting growth of the target species independently of their synergistic partners. This will domesticate these species and make them available to clinical research programs. We successfully conducted a concept study using environmental consortia, and utilize these experiences here for the purposes of oral microflora research. We note that all methodologies used and developed here are general in nature, and will be readily applicable to studies of other compartments of the human microbiome. We will share these technologies with the community, and make strains obtained here available to others to further progress in oral disease control and prevention. PUBLIC HEALTH RELEVANCE: One of the most important and intriguing observation in microbiology is that only few species grow in the lab. For the most part, the humane microbiome, including oral microflora, remains uncultivated and inaccessible for either basic or clinical research. In this project, we will develop two novel, and general, approaches based on single cell cultivation as well as co-cultivation of synergistic species, which will lead to isolation and domestication of novel species of clinical importance.